Observing the Effect of Polarity in the

Separation of Pigments Anish PrasannaJeremy Rubin

Aradhana VyasBlock A

Background• Used to separate mixtures

• Multiple types

• Developed in the early 1900s

Problem

• Determine the best isopropyl alcohol to water ratio

• Polarity-chromatographic separation relationship

Why our project is important• Chlorophyll often hides other pigments

• Is a sensitive method of detection

• Forensic Science

Paper Chromatography Introduction• Chloroplast Pigments

o Chlorophyll a and bo Anthocyanino Carotenoids

• Chromatographic Separation

http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography

Basic Chromatography Terms• Mobile Phase

• Stationary Phase

• Band Broadening Theory

• Solvento Solvent Fronto Solution

http://cellbiologyolm.stevegallik.org/aminoacids/page4

Basic Chemistry• Molecular Polarity

o Hydrogen Bondingo Dipole-Dipole Forceso Dispersion Forces

• Solubility• Capillary Action• Cohesion/Adhesion

http://www.science.uwaterloo.ca/~cchieh/cact/c123/intermol.html

Ion Dipole H Bond Dipole- Dipole

Hexane Dipole- Induced Dipole Dispersion

Chemistry-Van Deemter Equation

• H=A+B/u+Cuo Measures efficiency of

chromatographic separationo H=Plate Heighto u=Velocity of Mobile Phaseo A=Eddy Diffusiono B/u=Longitudinal Diffusiono Cu=Mass Transfer

http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography

Hypothesis and Null Hypothesis

• Hypothesiso 3:1 solution will provide best

separation Isopropyl alcohol-greatest

dispersion forces Lowest polarity Polar compounds-short distances Nonpolar compounds-long

distances• Null Hypothesis

o No relationship or relevance between polarity and separation of pigments

Independent and Dependent Variables

Independent Variable• Ratio of 1 M isopropyl alcohol to 1 M

watero 1:1o 3:1o 3:2

Dependent Variable• R(f) values of each pigment

o no units• Number of pigments

Red Leaf Extract-Control• Used as a standard

• Helped determine procedure

• Data collection

Materials Per Trial

• 3 test tubes• 3 mL of cranberry extract• 3 strips of chromatography paper • Pipette and pipette pump• 18.5 mL 1 M C3H8O• 11.5 mL 1 M H2O• Parafilm• Pencil• 3 50 mL Erlenmeyer Flasks• Ruler

Experimental Setup• Preparation of Isopropyl Alcohol/Water

Solvent

• Preparation of Chromatogram

• Data Collection R(f)

Determining the Color of Anthocyanin

Done through the Dynamic Model (STELLA)

Total Data Collected• Cranberry Extract

o 10 trials 1:1 ratioo 11 trials 3:1 ratioo 9 trials 3:2 ratio

• Red Leaf Extract (Control)o 4 trials 1:1 ratioo 2 trials 3:1 ratioo 5 trials 3:2 ratio

Sample Calculation R(f) Value• Distance traveled by solvent front

d(s)=10.7• Distance traveled by compound

d(c)=8.0036o R(f)=d(c)/d(s)

=8.0036/10.7 =0.748

• Average Pigments per chromatogram• Difference in R(f) values between

pigments o Calculated through median and

difference functions

Data Represented Through Median • Average number of pigments per ratio

(median)• Average R(f) value of pigment number

(median)

Average Number of Pigments per Chromatogram

Difference in R(f) Values Between Pigments (Cranberry)

Difference in R(f) Values Between Pigments (Red Leaf)

Data Analysis• Trends

o 3:2 ratio solvent resolved most pigments

o Red leaf extract separated more pigments than cranberry extract

o Pigments 1 and 2 have greatest difference in R(f) values

o 3:2 ratio created largest differences in R(f) values between chloroplast pigments

Problems Encountered

• Recording R(f) values from a chromatogramo Distinguishing between two bands

o Determining bands

o Determining the furthest extent of the solvent front

• Determining when a chromatogram is finishedo Remaining solvent

How Problems Were Overcome • Define standards for measuring R(f)

valueso Measure to center of color bando Solvent front-wetness of

chromatogramo Bands-variation in color

• Excess solvent in tubes-wait ten minutes for notable changes

Conclusionso 3:2 solvent mixture provided the most

effective separation Largest difference in R(f) values Most pigments separated on

chromatogramo Hypothesis is refutedo Relationship between polarity and

chromatographic separation As the ratio of dispersion forces and

dipole-dipole becomes closer, the greater the separation of pigments

Future Improvements• Create extract

• More trials

• Producing clearer resultso UV lampo Spectrophotometer o Longer chromatograms

Questions• What are three of the most common

pigments in plants?

• How does the polarity of the pigments determine how far they will travel up the chromatogram?

• By looking at the R(f) values of each pigment, how do you know when effective separation has been achieved?